Method and probes for the detection of a tumor specific fusion protein

ABSTRACT

This invention relates to the detection of fusion proteins. The invention provides a set of at least a first and a second molecular probe, each probe provided with a dye wherein the dyes together allow energy transfer, at least one probe provided with a reactive group allowing juxtaposing at least the first and second probes wherein the reactive group allows to modulate of juxtaposing the probes such that there is an increased likelihood of energy transfer between the dyes. A method is provided which permits detecting the presence of a fusion protein in a cell at the single cell level.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of PCT International PatentApplication No. PCT/NL2003/000776, filed on Nov. 6, 2003, designatingthe United States of America, and published, in English, as PCTInternational Publication No. WO 2004/042398 A1 on May 21, 2004, whichapplication claims priority to European Patent Application No.02079666.0 filed Nov. 7, 2002, the contents of the entirety of each ofwhich are hereby incorporated herein by this reference.

TECHNICAL FIELD

This invention relates to the detection of, among others, tumor-specificfusion proteins. More specifically, the invention relates to techniquesthat indicate the presence of chromosomal translocations by detectingthe presence of a fusion protein at the single cell level. In thediagnosis of various types of cancer, such as leukemias, lymphomas andsolid tumors, chromosome aberrations are frequently used forclassification into prognostically relevant subgroups.¹ Many of thesechromosome aberrations result in fusion genes, i.e., aberrantly coupledgenes coupled via the upstream part of one gene to the downstream partof the other gene, or vice versa. Fusion genes can be transcribed intofusion gene transcripts and translated into fusion proteins. Generally,fusion proteins play an important role in the oncogenetic process. Sofar, more than a hundred different fusion genes and fusion proteins havebeen described in various types of cancer.²⁻⁵

BACKGROUND

The term “cancer” comprises a heterogeneous group of neoplasms, in whicheach type has its own characteristics when considering its malignantpotential and its response to therapy. It goes without saying thataccurate diagnosis and classification of the various cancer types ispre-eminent in helping to select the most effective therapy.Furthermore, a diagnostic method allowing the detection of small numbersof malignant cells in a high background of normal cells during therapyis essential for evaluating treatment effectiveness and for anticipatingan impending relapse.

Chromosomal translocations can be detected by a wide array oftechniques, most of which entail modern biomolecular technology.Cytogenetic techniques include conventional chromosomal bandingtechniques (karyotyping) and fluorescence in situ hybridization (FISH)which uses fluorescently labeled probes. Polymerase chainreaction-(PCR—) based strategies can be used to detect fusions ofchromosomal breakpoints as can be found in chromosomal translocations,inversions and deletions using primers located at each side of thebreakpoint. DNA amplification can only be used for chromosomeaberrations in which breakpoints cluster in a small area. In most cases,breakpoints spread over large intronic regions, but severaltranslocations, inversions and deletions give rise to fusion genes andfusion transcripts suitable for PCR amplification after a reversetranscription step (RT-PCR).

Most commonly used techniques aimed at detecting specific Chromosomalaberrations involve analysis at the chromosomal or nucleic acid (DNA orRNA) level. An advantage of such genetic fusion markers is their directinvolvement in oncogenesis. Accordingly, their presence is constant allover disease evolution. However, a major drawback of fusion markersrelates to the fact that variations in the level of gene transcriptionand/or gene translation during the disease and particularly duringtherapy cannot be excluded. Thus, variations in expression of a fusiongene transcript or a fusion protein make it difficult to correlate thelevel of detection of the marker to the amount of malignant cells. Thisimplies that detection of a fusion gene product is preferably performedat the protein level in individual cells.

A fusion protein comprises parts of at least two proteins thatcorrespond to, and were originally transcribed by and translated fromthe originally separated genes. Fusion proteins are uniquelycharacterized by a fusion point, where the two proteins meet. Fusionpoints are often antigenically exposed, comprising distinct epitopeswhich sometimes can be immunologically detected.

Initially, attempts were made to raise fusion-protein specificantibodies by generating antibodies against a peptide corresponding tothe joining region of a fusion protein. This approach has rarely beensuccessful, mainly because of the fact that it is difficult to findimmunological reagents that are exclusively reactive with a fusionprotein and not with the non-fusion proteins that are normally producedin a cell. If fusion-specific antibodies were obtained, they weregenerally not applicable to fluorescence microscopy or flowcytometry.⁶⁻⁸ For example, the ERP-FP1 antibody against the BCR-ABLfusion protein works well in Western blotting procedures but is notsuccessful in microscopic studies on human BCR-ABL positiveleukemias.^(6,7) Moreover, considering the large variation withinindividual rearrangements seen in chromosomal translocations anddepending on the localization of the breakpoint within the non-aberrantgene (even when the translocations occur within the same two genes)wherein different fusion proteins can be generated, it is deemed likelythat within each separate case of fusion proteins, new fusion pointsarise. Detection of fusion proteins by specific immunologic detection ofthe fusion-point epitope of the fusion protein has therefore never beenwidely applicable.

An alternative method for the specific detection of fusion proteinsinvolves the application of a so-called catching antibody whichrecognizes one part of a fusion protein and a labeled detection antibodywhich recognizes an other part of a fusion protein. In such a system, acatching antibody is bound to a solid support layer, such as an ELISAplate or a dipstick. A catching antibody may also be immobilized ontobeads that can be analyzed by flow cytometry.⁹ Following incubation of acatching antibody with a cellular lysate suspected of containing thefusion protein, bound fusion protein is detected by a labeled detectionantibody. Although elegant and easy to perform, a catching/detectionantibody system cannot be applied practically to detect an intracellularfusion protein without disrupting the cell integrity. Mosttumor-specific fusion proteins are localized intracellularly, e.g.nuclear transcription factors, or signaling molecules which reside inthe cytoplasm or which shuttle between the cytoplasm and the nucleus.Thus, a catching/detection antibody system does not allow detection ofan intracellular fusion protein at the single cell level.

Co-localization of two differentially labeled antibodies against twodifferent parts of a fusion protein could in theory prove the presenceof a fusion protein in a single cell. However, full proof ofco-localization requires analysis by confocal laser scanning microscopy(CLSM). Even then it is generally not straightforward to evaluateco-localization of two antibodies because the recognized normalproteins, that are derived from the normal genes on the unaffectedchromosomes, can cause a background staining which interferes with thedetection of the fusion protein. Further, CLSM has the greatdisadvantage that it requires a specialized and well-equipped laboratoryand trained and highly skilled personnel. Such a time-consuming andhighly specialized technique is not desirable for routine diagnosticprocedures e.g. in a clinical setting.

All of the above indicate that there is a specific need for an improvedmethod to detect a fusion protein, which can preferably be used in aclinical laboratory. Particularly challenging is the detection of anintracellular fusion protein at the single cell level.

SUMMARY OF THE INVENYION

The invention provides the insight that fluorescence resonance energytransfer (FRET) technology can be used to detect the presence of afusion protein. The invention provides a method for detecting thepresence of a fusion protein in a cell using a set of at least a firstand a second molecular probe, each probe capable of recognizing abinding site positioned at opposite sides of the fusion region of thefusion protein, each probe provided with a dye wherein the dyes togetherallow energy transfer, comprising providing a set of probes, providing asample comprising a cell, contacting the sample with the probes underconditions that allow juxtaposing the probes on the fusion protein,removing any unbound and any non-specifically bound probe and detectingjuxtaposition of the probes via FRET to determine the presence of thefusion protein.

Also provided is a set of at least a first and a second molecular probe,each probe provided with a dye wherein the dyes together allow energytransfer; at least one probe provided with a reactive group allowingjuxtaposing the at least first and second probe, wherein the reactivegroup allows to modulate juxtaposing the probes such that there is anincreased likelihood of energy transfer between the dyes. According tothe invention, a molecular probe is capable of specifically binding to abiological molecule of interest via its so-called binding domain.Following binding of a at least a first and a second probe to a moleculeof interest via the binding domain, a reactive group can be used tomodulate juxtapositioning. A reactive group has no or a minimal tendencyto compete with the binding domain for binding to a molecule ofinterest. Herewith, a set of probes of the invention is distinguishedfrom known sets of antibody probes that are clustered or juxtaposed bythe mere binding to one antigenic molecule or complex. A reactive grouppreferably remains available for modulating the spatial organization ofjuxtaposed probes after the probe is bound to a molecule of interest. Inone embodiment, the molecule of interest is a protein, preferably afusion protein, more preferably an oncogenic fusion protein.Particularly preferred is a set of a first and a second molecular probewherein each probe is capable of recognizing and binding to a bindingsite (epitope) positioned at opposite sides of the fusion region of thefusion protein. Of course, when using a set of probes wherein each probebinds to a different epitope of a molecule of interest (e.g. epitopes atthe C- and N-terminal side of the fusion region of a fusion protein),the different epitopes should not interact with each other in either aninter- or intramolecular fashion because this would obviously interferewith probe binding. Different probes within a set of probes aretherefore capable of binding to different, essentially non-interactingepitopes. This is unlike the situation described in WO 01/75453 relatingto methods for detecting an entity by virtue of two probes (reporters),wherein the two probes may bind to the same target site on the entity,either substantially simultaneously or sequentially, or to differenttarget sites. The reporters/probes of WO 01/75453 may be used fordetecting a chimeric fusion protein. It is mentioned that one reporterpreferably binds an SH2 domain and the other reporter binds to anSH2-binding site, i.e. the probes of WO 01/75453 preferably bind tointeracting epitopes. Such probes and detection methods are clearlydistinct from the present invention because a FRET-based method asprovided herein would simply not work when using a set of probes whereindifferent probes are directed against either identical or interactingepitopes. Moreover, none of the probes of WO 01/75453 is provided with areactive group allowing juxtaposing the probes.

The present invention provides a diagnostic kit comprising a set ofprobes according to the invention and a method using a set of probes fordetecting the presence of a fusion protein in the diagnosis and/orclassification of a disease as well as before, during and aftertreatment of a disease to evaluate the effectiveness of the treatment.

Also provided is a method for producing a probe set according to theinvention comprising contacting each probe with a dye to form aconjugate between the probe and the dye and purifying the conjugate,further comprising contacting at least one probe with a reactive groupor a derivative thereof to form a conjugate between the probe and thereactive group and purifying the conjugate.

Fluorescence resonance energy transfer (FRET) is a distance-dependentinteraction between the electronic excited states of two dye moleculesin which a “donor” molecule, after excitation by a light source,transfers its energy to an“acceptor” molecule. In general, the donor andacceptor dyes are different, in which case FRET can be detected by theappearance of sensitized fluorescence of the acceptor or by quenching ofdonor fluorescence. When the donor and acceptor dyes are the same, FRETcan be detected by the resulting fluorescence depolarization. Energytransfer occurs when the emission spectrum of the acceptor overlapsignificantly. To achieve resonance energy transfer, the donor mustabsorb light and transfer it through the resonance of excited electronsto the acceptor. ¹⁰⁻¹³ FRET is usually based on the interaction betweendonor and acceptor dyes that are both fluorescent. However,non-fluorescent acceptor dyes can also be used. Nonfluorescent acceptordyes can be advantageous because they eliminate the backgroundfluorescence that results from direct (i.e., nonsensitized) acceptorexcitation. In the present invention, it is possible to monitorjuxtaposed probes on a fusion protein using a fluorescent donor dye anda nonfluorescent acceptor dye. Specific binding of a set of probes tothe native proteins, e.g. proteins A and B, will give a basalfluorescence signal. Upon close juxtapositioning of a set of probes on aA-B fusion protein, FRET between the probes will quench the donorfluorescence. Rather than measuring an increase in acceptorfluorescence, use of a nonfluorescent acceptor involves measuring adecrease in donor fluorescence. Generally speaking, detection of adecreased signal is less sensitive compared to detection of an increasedsignal. Therefore, a method according to the invention is preferablypracticed using a fluorescent donor and a fluorescent acceptor dye.

For energy transfer to take place, the fluorescence emission wavelengthof the donor must be lower than the excitation wavelength of theacceptor; that is, the process must be energetically “downhill”.Sufficiently close juxtaposition of the two dyes, generally closer than100 Δngstrom but preferably closer than 50 Δngstrom, is essential forenergy transfer between the donor/acceptor pair. One Δngstrom, a metricunit of length, is equal to 0.1 nanometer or 10⁻¹⁰ meter. The FRETenergy transfer efficiency is inversely proportional to the sixth powerof the distance between the donor and the acceptor. The insight isprovided that, due this high sensitivity to distance, FRET is especiallysuitable to detect the juxtaposing of two different dye-conjugatedprobes on a fusion protein.

In a preferred embodiment of the invention, a probe set comprises a setof at least two dye-conjugated antibodies, each antibody capable ofrecognizing a binding site positioned at opposite sides of the fusionregion of a fusion protein. A suitable antibody comprises a conventional(poly- or monoclonal) or a synthetic antibody or a binding fragmentfunctionally equivalent thereto, such as a Fab′, Fab, a single chain Fvfragment, a diabody (a single chain Fv dimer) and the like. For example,a chimeric fusion protein A-B can be detected via FRET using a set ofdye-conjugated probes, e.g. an anti-A antibody and an anti-B antibody.In a preferred embodiment, a sample is contacted with two antibodies,one against domain A and the other against domain B of a fusion proteinto detect the presence of an A-B fusion protein in a cell sample. Oneantibody is labeled with a FRET donor dye and an other with a FRETacceptor dye. Only when domain A is in close proximity to domain B, e.g.when both are part of the same protein molecule, the two antibodiesbecome sufficiently close together (“juxtaposed”) which allows thedonor/acceptor pair to induce a detectable FRET fluorescence signal.

Simultaneous reactivity of more than one different antibodies with thesame protein molecule needs recognition of two different binding sitesor epitopes that are sufficiently separated in order to prevent sterichindering of the antibodies. For example, simultaneous application of anantibody against the variable (V) domains and an antibody against theconstant (C) domains of T-cell receptor (TCR) molecules on the cellsurface of a T-lymphocyte gives no reliable and reproducible results.However, simultaneous application of V domain antibodies and an antibodyagainst the CD3 molecule, which is closely associated with the TCRmolecule, yielded excellent staining results in both flow cytometry andmicroscopy.¹⁴ These data suggest that the distance between two epitopeson the same protein should preferably be more than approximately 80Δngstrom to be recognized simultaneously.

Co-localization of two dye-conjugated antibodies against different partsof the same fusion protein is sometimes not sufficient for the requiredFRET energy transfer. A complete antibody is a large Y-shaped proteinmolecule, ˜150 kDa in size, made up of two heavy chains and lightchains. Owing to the length of an antibody molecule (300 to 400Δngstrom) and the flexibility of the hinge region, juxtaposed antibodymolecules can bridge a relatively large distance.¹⁵ Whereas closelyjuxtaposed FRET probes are in general sufficient for obtaining a FRETsignal, it may be advantageous to stabilize and/or enhance juxtaposingtwo probes in order to increase FRET efficiency. For example, the sizeof a probe or a dye might interfere with FRET analysis via negativesteric effects. Also, the flexibility of an antibody may decrease theprobability of FRET occurrence between a pair of FRET dyes that areconjugated to antibody probes. When preparing a dye conjugate, like afluorescent probe, it is in general not possible to control the site ofconjugation. For example, in case of antibody conjugation, a dye moietymight become attached to different parts of the antibody molecule.Depending on the site of dye-conjugation, the spatial orientation ofdyes on probes can be favorable or unfavorable for FRET energy transferefficiency i.e. dyes attached to probes need not necessarily be withinenergy transfer distance of each other.

Surprisingly, the invention provides the insight that juxtaposing a setof probes can be modulated in order to increase the probability of FRETenergy transfer between a pair of dyes, by providing at least one probewith a reactive group. The invention provides a set of at least a firstand a second molecular probe, each probe provided with a dye wherein thedyes together allow energy transfer; at least one probe comprising areactive group allowing juxtaposing at least first and second probewherein the reactive group allows modulation of juxtaposing the probessuch that there is an increased likelihood of energy transfer betweenthe dyes. Use of such a probe set allows to detection of juxtaposedprobes with an improved sensitivity compared to use of probes notcomprising any reactive groups.

In the present context, the term “reactive group” refers to a moietythat allows modulating the spatial organization of FRET dyes such thatthere is an increase in the probability of energy transfer to occurand/or an increase in energy transfer efficiency. The spatialorganization refers to both the distance between the dyes as well as totheir relative orientation. Modulating the spatial organization includesadjusting and stabilizing the spatial organization of dyes. One of theprimary conditions for energy transfer to occur is that donor andacceptor molecules must be in close proximity, typically 10-100 Δ. In apreferred embodiment, a reactive group allows juxtaposing the dyeswithin a distance of 100 Δ of each other, more preferably within 50 Δ ofeach other but most preferably within a distance of 20 Δ of each other.It is therefore preferred that a reactive group is small, e.g., smallerthan 10 kiloDalton (kD),more preffered, smaller than 5 kDa, even morepreffered, smaller than 2 kDa or most preffered, smaller than 1 kDa. Forexample, a reactive group is biotin.

As the, a reactive group allows modulating juxtaposed probes such thatthere is an increased likelihood of energy transfer between dyes bydirectly interacting with another probe. For example, a reactive groupof a first probe binds to a part of a juxtaposed second probe to form astable complex between the probes in a spatial orientation that isfavorable for FRET to occur. As mentioned above with respect to the siteof dye conjugation, it is often not possible to selectively modify aprobe with a reactive group at a defined site. The site of modificationis mainly determined by the presence and accessibility of a certainresidue via which a reactive group is conjugated to a probe, e.g. viaprimary amines or via thiol groups. Thus, an antibody probe may containa reactive group at either the constant and/or the variable region ofthe immunoglobulin. It is conceivable that not every site is equallysuitable for interacting with a second probe e.g. due to sterichindrance. Therefore, it is preferred that a probe is provided with amultiplicity of reactive groups to statistically increase its capacityto interact with another probe. For example, a probe is provided withtwo or three or even five reactive groups.

Provided herein is a method for detecting the presence of a fusionprotein in a cell using a set of at least a first and a second molecularprobe, each probe capable of recognizing a binding site (via its bindingdomain) positioned at opposite sides of the fusion region of the fusionprotein, each probe further provided with a dye wherein the dyestogether allow energy transfer, at least one probe provided with areactive group allowing modulation of juxtaposing the at least first andthe second probe such that there is an increased likelihood of energytransfer between the dyes, comprising providing a set of probes,providing a sample comprising a cell, contacting the sample with theprobes under conditions that allow juxtaposing the probes on the fusionprotein, removing any unbound and any non-specifically bound probe anddetecting juxtaposition of the probes via FRET to determine the presenceof the fusion protein. In case a first probe can interact directly withat least a second probe, it is preferred to contact the sample with eachprobe in consecutive steps with extensive intermittent washingprocedures to avoid self association between probes. For example, asample is contacted with probe A, comprising a reactive group, to allowrecognition of and binding to one part of a fusion protein. Next, anyunbound and any non-specifically bound probe A is removed by repeatedwashing steps. Subsequently, the sample is contacted with probe Breactive with another part of the fusion protein under conditionsallowing juxtaposing probe A and B on the same fusion protein. Alsohere, any unbound and any non-specifically bound probe B is preferablyremoved by repeated washing steps. In one embodiment of the invention, areactive group of probe A interacts with at least a juxtaposed probe Bto enhance and/or stabilize the spatial orientation of the dyes presenton the probes such that there is an increased likelihood of energytransfer between them. Although this method can be used to detect thepresence of a fusion protein, it shall be clear that such a procedure,involving multiple separate contacting and washing steps, can be ratherlaborious and time-consuming. Moreover, if probes are capable ofdirectly interacting with each other, a significant background stainingcan be expected caused by probes binding to the domains on the normalproteins that are derived from the normal genes instead of the fusiongene. In the example above, a reactive group of probe A which is boundto the native protein A might recruit and interact with probe B. Also,if not all unbound probe A is efficiently removed, an unwantedinteraction between probe A and B can occur upon contacting the samplewith probe B. Both events may result in a detectable energy transfersignal, despite the fact that probe B is not juxtaposed to probe A on afusion protein.

Thus, in a preferred embodiment of the invention, a reactive group of afirst probe is not directly or immediately reactive with a second probein order to avoid self association of the probes. This is advantageousfor an optimal recognition of a fusion protein by each probe and forjuxtaposing the probes on the fusion protein. Moreover, it avoidsuntimely energy transfer to occur between directly connected ormultimerized probes and decreases an aspecific background signal. Thisis important to ensure that an energy transfer signal truly reflectsjuxtaposed probes.

The invention provides the insight that, if a reactive group of a firstprobe is not reactive with at least a second probe in order to avoidself association of the probes, a so-called “bridging” substance may beused to mediate an interaction between the probes, allowing to modulatejuxtaposing the probes such that there is an increased likelihood ofenergy transfer between the dyes on the probes. A substance may be anykind of compound capable of binding to or modifying a probe, a reactivegroup and/or a dye to modulate the spatial organization of dyes onjuxtaposed probes such that it is favorable for FRET. Preferably, asubstance allows juxtaposing the dyes within a distance of 2 to 100Δngstrom of each other. The substance is preferably added to a samplefollowing binding of dye-conjugated probes to a target fusion protein,in an amount effective to modulate the spatial organization of the dyeson juxtaposed probes. Advantageously, the substance binds to a reactivegroup with a high specificity and a high affinity. Also, it is preferredthat such a substance is relatively small so that the bridging substanceonly minimally affects the distance between a pair of dyes and therelative orientation of a pair of dyes.

In a preferred embodiment, a method is provided for detecting thepresence of a fusion protein in a cell using a set of at least a firstand a second molecular probe, each probe capable of recognizing abinding site positioned at opposite sides of the fusion region of thefusion protein, each probe further provided with a dye wherein the dyestogether allow energy transfer, at least one probe provided with areactive group allowing to modulate juxtaposing at least first and thesecond probe such that there is an increased likelihood of energytransfer between the dyes, wherein a reactive group of the first probeis not directly reactive with the second probe, comprising providing aset of probes providing a sample comprising a cell, contacting thesample with the probes, under conditions that allow juxtaposing theprobes on the fusion protein, removing any unbound and anynon-specifically bound probe, contacting the probes with a substancecapable of linking at least a reactive group of the first probe to thesecond probe and detecting juxtaposition of the probes via FRET todetermine the presence of the fusion protein.

A method using a probe set at least one probe comprising a reactivegroup wherein probes do not directly interact and require a bridgingsubstance has several advantages. First, an improved specificity andreduced background staining can be achieved compared to a method usingprobes which can directly interact. After all, for a reactive group toexert its effect via a bridging substance, probes need to be in a closejuxtaposition of each other prior to the addition of the substance i.e.resulting from binding of one probe adjacent to another probe on thesame fusion protein. Second, the procedure is fast and easy because noseparate contacting/washing steps are required for each individualprobe. Thus, it permits to contact a sample with a mixture of probes alltogether in a single action. Likewise, any unbound and anynon-specifically bound probes can be removed simultaneously. Muchpreferred, as exemplified herein in the detailed description, is a setof at least a first and a second molecular probe, each probe providedwith a dye wherein the dyes together allow energy transfer; each probeprovided with a reactive group. A substance is preferably capable ofbinding, or “bridging”, at east two reactive groups. In a preferredembodiment, each probe within a set of probes is provided with the samereactive group. Also, each probe within a set of probes may be providedwith a different reactive group but having the same reactivity. Thisallows the use of one type of bridging substance having at least twoidentical binding sites for a reactive group.

In a preferred embodiment, a probe is provided with more than onereactive group, enabling the probe to interact with more than onemolecule of bridging substance. Providing a probe with more than onereactive group will theoretically increase the likelihood of aninteraction between the probe and a bridging substance. Furthermore, forthe ease of practicing the present invention, a suitable reactive groupor a derivative thereof is commercially available and can be easily andefficiently attached to a probe.

In accordance with the present invention, a particularly interestingreactive group is biotin, with avidin or streptavidin being aparticularly suitable bridging substance. Avidin is an egg-white derivedglycoprotein with a molecular weight of about 68000 daltons and adiameter of 8 to 10 Δngstrom. It consists of four identical subunitchains. One avidin or streptavidin molecule can bind four molecules ofbiotin. Avidin has an extraordinarily high affinity(affinity constant>10¹⁵ M−1) for biotin. This high affinity assures the user of a rapidlyformed and stable complex between avidin and the biotin-labeled probes.The protein streptavidin, produced by the bacterium Streptomycesavidinii, has a structure very similar to avidin, and also binds biotintightly. It often exhibits lower non-specific binding, and thus isfrequently used in place of avidin. Once a biotin-avidin complex forms,the bond is essentially irreversible. The biotin-avidin system is widelyused and has proven to be very useful in the detection and localizationof antigens, glycoconjugates, and nucleic acids by employingbiotinylated antibodies, lectins, or nucleic acid probes. As the, areactive group with such a small size is advantageous for achieving aclose distance between a dye pair. Biotin is a vitamin with a molecularweight of only 244 daltons. Also, many biotin molecules can be coupledto a protein, enabling the biotinylated protein to bind more than onemolecule of avidin. Avidin, streptavidin and biotin are available frommany commercial sources. Various standard procedures for preparingbiotin-conjugates are known to those skilled in the art, most of whichcan be completed within a day. Moreover, commercial biotinylation kitsare available that contain all the necessary components for proteinbiotinylation.

If a set of probes is used wherein each probe is provided with adifferent reactive group, a suitable substance comprises a moleculecapable of binding at least one of each reactive group. Alternatively,such a binding substance comprises a complex of at least two moleculesthat can be covalently or non-covalently attached to each other, whereineach molecule is capable of binding to a reactive group.

The invention provides a method for detecting a fusion protein at thesingle cell level using of a set of probes according to the invention,each probe capable of binding to a binding site positioned at oppositesides of a fusion region of the fusion protein via the binding domain ofthe probe i.e. one probe is directed against a protein fragmentcomprising the N-terminal fragment of a fusion protein, and an otherprobe is directed against a protein fragment comprising the C-terminalfragment of the same fusion protein. A fusion protein comprises any kindof proteinaceous substance which is formed after transcription andtranslation of a fusion gene. A fusion gene comprises one part of one ormore genes combined with another gene or a part derived thereof. Afusion protein may be the result of a chromosomal translocation,inversion or deletion. In a preferred embodiment, a method provided isused to detect a tumor-specific fusion protein. A fusion protein may bean endogenously expressed protein or it may be the result of geneticengineering. Fusion proteins in malignancies which can readily bedetected using a method according to the invention include but are notlimited to those listed in Table I.

It is of great relevance to note that the present method does notrequire disruption of the cell integrity, e.g. the preparation of a celllysate, to detect the presence of an intracellular fusion protein.Preservation of the morphology integrity of a cell permits analysis atthe single cell level, for example by flow cytometry or fluorescencemicroscopy. Detection of a FRET signal by flow cytometry offers theability to perform rapid, multiparametric analysis of specificindividual cells in a heterogeneous population. The main advantage offlow cytometry is that it directly gives quantitative data and that itis very rapid (results can be obtained in a few hours).

The method provided in the present invention allows detection of afusion protein at the single cell level. In a preferred embodiment, themethod provided is used to detect an intracellular protein at the singlecell level. When detecting an intracellular fusion protein, a samplecomprising a cell is treated so as to obtain a permeabilization of thematerial and a preservation of the morphology. The preferred treatmentis one which fixes and preserves the morphological integrity of thecellular matrix and of the proteins within the cell as well as enablesthe most efficient degree of probe, e.g. antibody, penetration.

Unlike for example a “catching/detection” antibody method, which canessentially only be applied to detect the presence of a fusion proteinat the cell surface or in a cell lysate, the present method allowsgating of subset of cells that are present in a mixture of cells viaimmunophenotypic characteristics. Consequently, the method providedherein permits the detection of a fusion protein in a rare population ofmalignant cells in a large background of normal cells. This isespecially advantageous for detecting low frequencies of fusion-positivecells like in the case of detection of minimal residual disease (MRD)during or after treatment for evaluation of treatment effectiveness. Inpreferred embodiment, the method provided includes multiparameter flowcytometry to identify and/or isolate single cells to detect the presenceof a fusion protein at the single cell level. All that is required forpracticing the method provided is a flow cytometry facility.Importantly, the procedure can be performed in routine laboratories bypersonnel with ordinary skills.

More than a hundred different fusion genes and fusion proteins have beendescribed in various types of cancer. As the, the method provided allowsto discriminate between the presence of normal proteins and an aberrantfusion protein at the single cell level. Theoretically, two antibodiesrecognizing two different domains of a fusion protein can cause abackground staining by binding to the domains on the normal proteinsthat are derived from the normal genes instead of the fusion gene.However, generally only one of the two normal proteins reaches adetectable expression level in a target cell population, as defined bycell surface and/or intracellular markers. Furthermore, the normalproteins and the fusion protein often differ in their intracellularexpression pattern, frequently resulting in a different subcellularlocalization.^(16, 17) This implies that coincidental co-localization ofthe two different normal proteins is unlikely to occur at a significantlevel in the target cell population. In particular, coincidentaljuxtaposing probes sufficient for a FRET signal will be rare in normalcells, if this occurs at all.

Provided herein is a method for producing a set of at least a first anda second molecular probe, each probe provided with a dye wherein thedyes together allow energy transfer; at least one probe provided with areactive group allowing juxtaposing the first and second probe,comprising contacting each probe with a dye to form a conjugate betweenthe probe and the dye and purifying the conjugate, further comprisingcontacting at least one probe with reactive group or a derivativethereof to form a conjugate between the probe and the reactive group andpurifying the conjugate. The Förster radius (R₀) is the distancecorresponding to 50% energy transfer efficiency and it characterizeseach donor/acceptor pair. Its value is generally between 30 and 60Δngstrom. In the present context, the term “dye ” refers to asubstituent that, in concert with another dye, can be used for energytransfer analysis, such as FRET analysis. As mentioned above, FRET isusually based on the interaction between donor and acceptor dyes thatare both fluorescent. In one embodiment, the invention uses a set ofprobes wherein at least one of the dyes is a fluorochrome. However, anonfluorescent acceptor may also be used and FRET is detected byquenching of donor fluorescence. As said, detecting FRET by monitoring adecrease in donor fluorescence as a consequence of juxtapositionedprobes is often not as sensitive as detecting in increase in acceptorfluorescence. Thus, in a preferred embodiment, at least twofluorescently labeled probes are used to detect a fusion protein, as isexemplified in the detailed description. Examples of preferredfluorochromes are those suitable for analysis by conventional flowcytometry and include fluorescein labels, e.g. 5-(and 6-)carboxyfluorescein, 5- or 6- carboxyfluorescein, 6-(fluorescein)-5-(and6-) carboxamide hexanoic acid and fluorescein isothiocyanate, AlexaFluor dyes such as Alexa Fluor 488 or Alexa Fluor 594, cyanine dyes suchas Cy2, Cy3, Cy5, Cy7, optionally substituted coumarin, R-phycoerythrin,allophycoerythrin, Texas Red and Princeston Red as well as conjugates ofR-phycoerythrin and, e.g. Cy5 or Texas Red and members of thephycobiliproteins. Other dyes of interest are quantum dot dyes, whichcome in a nearly unlimited palette of colors. Extensive information ondonor/acceptor pairs suitable for energy transfer detection by flowcytometry can be found in Szollosi et al.¹⁸ Preferred combinations offluorochromes comprise those dyes used in the classical tandemconjugates, also referred to as duochromes.¹⁹

The method provided comprises providing a sample comprising a cell,whereby the sample is optionally subject to fixation andpermeabilization if an intracellular fusion protein is to be detected. Asample may comprise a primary cell that is obtained from a biologicalsample. A biological sample can be a body fluid sample including blood,serum, urine, bone marrow, cerebrospinal fluid (CSF), or saliva. It mayalso be a tissue sample or tissue homogenate. A sample comprises acultured cell that may be a cultured primary cell, for example, tumorcells obtained from a lymph node biopsy. Furthermore, a sample maycomprise a cultured cell from an established laboratory cell line, likea K562, KASUMI-1, REH or CEM cell line, which can be obtained from anumber of sources such as the American Type Culture Collection (ATCC;worldwideweb:atcc.org for an online catalog. The method provided issuitable for detection of the presence of an endogenous fusion protein,as well as a recombinant fusion protein, in a cell.

For analyzing a sample comprising a suspension of cells, it is preferredthat the sample is treated so as to obtain a preservation of themorphology of the material and permeabilization in order to ensuresufficient accessibility of a molecule of interest to a probe. The typeof treatment will depend on several factors, for instance on thefixative used, the extent of fixation and the type and properties of themolecule of interest. Fixation may be carried out with a fixative suchas formaldehyde.

For the detection of a fusion protein in primary cells, it is especiallyadvantageous to use an additional marker to define a target cellpopulation of interest. A number of important biological applications ininfectious diseases, MRD detection and monitoring, and gene therapytypically require the analysis and isolation of rare cells (e.g.haemopoietic stem/progenitor cells) from a large background. In oneembodiment of the invention, the method includes staining a sample forat least one cellular marker, like a cell surface marker or anintracellular marker, to define a target cell population within amixture of cells comprising contacting the sample with a compoundcapable of selectively binding to the marker. In a preferred embodiment,such a compound is directly tagged with a fluorescent dye. A suitablecompound comprises a fluorescently labeled antibody or a bindingfragment functionally equivalent thereto. Also, a compound capable ofselectively binding to a cellular marker can be used which can bedetected using a dye-conjugated secondary reagent (e.g. a fluorescentlylabeled secondary antibody). A cellular marker comprises any kind ofintracellular or membrane-bound marker which can be used to distinguisha subpopulation of cells in a mixture of cells. A mixture of cellscomprises living cells. It also comprises permeabilized and/or fixedcells. A cellular marker can be a cluster of differentiation (CD)antigen. CD markers are cell surface molecules of, among others,haemopoietic cells that are distinguishable with monoclonal antibodies.Haemopoietic cells comprise thymocytes, dendritic cells, Langerhans'cells, neutrophils, eosinophils, germinal centre B cells, folliculardendritic cells, plasma cells and bone-marrow cells. For example,suitable cellular markers comprise CD1, CD3, CD4, CD8, CD10, CD19, CD20,CD33, CD34 and CD117. Monoclonal antibodies directed against a largenumber of human CD markers can be obtained from various suppliers, suchas BD Biosciences or Ancell Immunology search Products, Bayport, USA.Often, antibodies are available that are directly conjugated with afluorochrome of choice e.g. CD10-PE or CD19-FITC, which is obviously apreferred choice to practice a method according to the invention.

In a preferred embodiment, a method is provided to identify and/orisolate rare single cells using multiparameter flow cytometry/cellsorting techniques and to further characterize these cells by thepresence or absence of a fusion protein of interest. Such a method isparticularly suited for application to a number of important problems inimmune system development, infectious diseases, cancer and gene therapy.Typically, prior to staining a cell sample with a probe set, cells arelabeled with at least one relevant dye-conjugated antibody according tostandard procedures in order to define a target cell population. Thechoice of dye should preferably, but not exclusively, aim at the usageof two or three dyes for immunophenotyping in addition to the FRET dyesfor detection of a fusion protein. For example, a FRET probe setaccording to the invention can be combined with another dye to mediateleukocyte subset gating via immunophenotypic characteristics, e.g. CD10,CD19 and CD20 to accurately define subsets of precursor-B-cells in bonemarrow, or CD1, CD4 and CD8 to define subsets of thymocytes, or CD84and/or CD117 to identify stem/precursor cell populations. As shownherein in the detailed description, the invention provides a method thatallows the detection of an intracellular fusion protein in a very smallsubset of cells, i.e. detection of MRD, which is essential forevaluating effectiveness of cancer treatment.

The invention provides a diagnostic test kit for detecting the presenceof a fusion protein in a cell comprising a set of probes according tothe invention For example, such a kit may be used for monitoring andquantification of malignant cells, e.g. leukemic cells, via thedetection of tumor-specific fusion protein-positive cells. Thediagnostic test kit provided herein is useful at the time of diagnosisas well as during and after treatment to evaluate the effectiveness ofthe applied cancer treatment protocol.

TABLE I Examples of fusion proteins in malignacies that can be detectedvia antibody-mediated FRET technology. Malignancy Chromosome aberrationFusion prrotein Precurso-B-ALL t(1;19)(q23;p13) E2A-PBX1t(4;11)(q21;q23) MLL-AF4 t(9;22)(q34;q11) BCR-ABL t(12;21)(p13;q22)TEL-AML1 Acute myeloid t(8;21)(q22;q22) AML1-ETO leukemiat(15;17)(q22;q21) PML-RARA inv(16)(p13;q22) CBFB-MYH11 Lymphomat(2;5)(p23;q35) NPM-ALK Ewing sarcoma t(11;12)(q24;q12) EWS-FLI1

DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic diagram of a fusion gene consisting of the upstream(5′) part of gene A and the downstream (3′) part of gene B. This A-Bfusion gene is transcribed into A-B mRNA and translated into a A-Bfusion protein.

FIG. 2. Schematic diagram of the principle of fluorescence resonanceenergy transfer (FRET) with fluorochrome X as donor dye and Y asacceptor dye. A. The acceptor dye Y will not be excitated by theemission light of the donor dye X, if the distance between X and Y istoo large. B. If the distance between the donor and acceptor dye issufficiently small (<80 Δngstrom but preferably<50 Δngstrom), theemission light of the donor dye X will excitate the acceptor dye Y.

FIG. 3. Schematic diagram of the A-B fusion protein recognized by a setof anti-A and an anti-B antibody probes. A. Probe A is conjugated withdonor dye X and probe B is conjugated with acceptor dye Y (see FIG. 2).Furthermore, both probes are conjugated with biotin as a reactive group.B. After incubation with antibody probes A and B, the probes can bebound together via incubation with avidin, provided that the two probesindeed recognize and bind to the same A-B fusion protein. Thisjuxtaposition of the two antibodies (stabilized by the biotin-avidinsystem) is detectable via the FRET principle (see FIG. 2).

FIG. 4. Example of FRET-mediated detection of the TEL-AML1 fusionprotein in ALL cells. A. Precursor B-ALL cells at diagnosis. Flowcytometric gating on ALL blast cells as defined by light scattercharacteristics (left), followed by gating on CD 19+ blast cells(middle), and evaluation of the presence of the TEL-AML1 fusion proteinwithin the CD10⁺/CD19⁺ ALL cells (right). B. Precursor B-ALL cellsduring follow-up. Flow cytometric detection of low frequencies ofTEL-AML1 positive cells (minimal residual disease) during follow-up forevaluation of treatment effectiveness. Only 3% of the CD10+ blasts waspositive for TEL-AML1 fusion protein, i.e. only 0.2% of totalleukocytes.

DETAILED DESCRIPTION OF THE INVENTION

As mentioned above, the present invention relates to a method fordetermining the presence of a fusion protein in a cell using a probeset. This method can be used to diagnose various types of cancer whichinvolve chromosomal translocations, inversions or deletions that giverise to a fusion gene. For example, approximately 35% of adult patientswith acute lymphoblastic leukemia (ALL) and chronic myeloid leukemia(CML) are associated with a specific chromosomal defect, a translocationbetween chromosomes 9 and 22 that creates the Philadelphia (Ph)chromosome. This translocation occurs at the site in the genome of aprotein tyrosine kinase named ABL, creating the abnormal BCR-ABL fusionprotein, a gene product of the in-frame fusion of the ABL gene withanother gene called BCR. Generally, fusion proteins play an importantrole in the oncogenetic process. For example, the kinase activity of ABKin the BCR-ABL fusion protein is activated and deregulated, driving theuncontrolled cell growth observed in ALL and in CML. When acutelymphoblastic leukemia is diagnosed in a patient, typically comprisingtraditional cytogenetics such as karyotype analysis for the Phchromosome, the total number of leukemia cells is approximated to 10¹¹to 10¹³. A majority of patients reach complete remission after about 5weeks of chemotherapy. Complete remission does not mean that theleukemic cells are totally eradicated from the body but that their levelis beyond the sensitivity level of classical cytomorphologic methods(e.g. 1 to 5%). At this time, up to 10¹⁰ malignant cells can stillremain in the patient. They represent the minimal residual disease(MRD). Detection of low frequencies of residual malignant cells allows alonger follow-up of the tumor burden during chemotherapy and thus,permits to better appreciate the sensitivity of leukemia cells totreatment. It is now established that the level of MRD represents apowerful prognostic factor for final outcome. Besides, the detection ofan increase of the MRD level enables to anticipate impending relapse.The method provided in the present invention allows to discriminatebetween the presence of normal proteins and an aberrant fusion proteinat the single cell level.

As an example of this method we will describe the preparation of a probeset for the detection of the TEL-AML1 fusion protein. Also described isa method using this probe set to detect the presence of TEL-AML1 fusionprotein in ALL cells at the time of diagnosis and during follow-up todetect the level of MRD.

EXAMPLE

Preparation of a Set of Probes

Preferably, a probe set according to the invention comprises a set oftwo fluorochrome-conjugated antibodies each antibody additionallyprovided with a reactive group. Methods of producing an antibody areknown to those skilled in the art. For example, to obtain a polyclonalantibody, a laboratory animal is immunized with an immunogen such as arecombinant protein or a synthetic peptide. The animal's immune responseis monitored by taking test bleeds and determining the titer of thereactivity. When appropriately high titers are obtained, blood iscollected from the animal and antisera are prepared.

Further fractionation of the antisera to enrich for antibodies reactiveto the protein can be done if desired. See e.g. Harlow et al.Antibodies. A Laboratory Manual, Cold Spring Harbor Publications, NewYork (1988). Monoclonal antibodies can be obtained by various techniquesknown in the art, for example by fusing spleen cells of immunized micewith a myeloma cell line by the addition of polyethylene glycol (PEG).Fused cells are cultured in a selection medium, e.g. medium containing amixture of hypoxanthine, aminopterin and thymidine. Fused cells thatsurvive in this selection medium are tested for the production of thedesired antibody (often by solid-phase immunoassay such as ELISA) and,if positive, the cultures are cloned so that there is only one cell ineach culture well. This produces a clone of cells from a singleprogenitor which is both immortal and a producer of monoclonal antibody.Antibodies obtained can be characterized using conventionalimmunodiagnostic techniques e.g., by Western blotting using lysates ofcells expressing a recombinant fusion protein or by ELISA.

Biotinylation of Antibodies

Biotin is typically conjugated to proteins via primary amines (i.e.,lysines). Usually, between three and six biotin molecules are conjugatedto each antibody. Dialyze or exchange over a column the antibody in 100mM carbonate, pH 8.4. Measure the antibody concentration after bufferequilibration. (For IgG, 1 mg/ml has an A₂₈₀ of 1.4). If the antibodyconcentration is less than 1 mg/ml, the conjugation will probably besub-optimal. If necessary, dilute the antibody to a concentration of 4mg/ml. Dissolve 10 mgs of biotin (N-hydroxysuccinimidobiotin, Pierce) in1 ml anhydrous DMSO (anhydrous dimethyl sulfoxide, Aldrich) immediatelybefore use. The reactive biotin molecule is unstable. Once the biotin issolubilized, it should be used immediately. Add biotin to give a ratioof 80 μg per mg of antibody; mix immediately. Wrap the tube in foil;incubate and rotate at room temperature for two hours. Remove theunreacted biotin and exchange the antibody into 10 mM Tris pH 8.2, 150mM NaCl, pHix (5 mg/ml pentachlorophenol in 95% ethanol (use as 10,000×,or 3-4 drops per liter) Sigma).

FITC Conjugation of an Antibody

FITC is a small organic molecule, and is typically conjugated toproteins via primary amines (i.e., lysines) of an immunoglobulin.Usually, between three and six FITC molecules are conjugated to eachantibody; higher conjugations can result in solubility problems as wellas internal quenching (and reduced brightness). Thus, an antibody willusually be conjugated in several parallel reactions to different amountsof FITC, and the resulting reagents will be compared for brightness (andbackground stickiness) to choose the optimal conjugation ratio. Theentire conjugation can be performed in about a half-day. The reactivefluorescein molecule, fluorescein isothiocyanate, is unstable. Once avial has been cracked and the FITC solubilized, it should be usedimmediately. Since single vials of FITC contain sufficient material for˜100 mgs of antibody, it is economical to perform multiple FITCconjugations on the same day.

1. Antibody Preparation

Dialyze or exchange over a column the antibody in 500 mM carbonate, pH9.5. Measure the antibody concentration after buffer equilibration. (ForIgG, 1 mg/ml has an A₂₈₀ of 1.4). If the antibody concentration is lessthan 1 mg/mL, the conjugation will probably be sub-optimal. Ifnecessary, dilute the antibody to a concentration of 4 mg/ml.

2. Covalent Conjugation

Dissolve 10 mgs (the entire contents of 1 vial; no need to weigh) ofFITC (Molecular Probes) in anhydrous DMSO immediately before use. AddFITC to give a ratio of 40-80 μg per mg of antibody; mix immediately.Wrap the tube in foil; incubate and rotate at room temperature for onehour. Remove the unreacted FITC and exchange the antibody into 500 mMcarbonate, pH 9.5 by gel filtration or dialysis.

3. Characterization of the Conjugate

Determine F/P and protein concentration by measuring the absorbance at280 and 495 nm. IgG: 1 mg/ml has an A(280) of 1.4; mw=150,000. IgM: 1mg/ml has an A(280) of 1.2; mw=900,000. Fluorescein: 1 mM has an A(495)of 68 and an A(280) of 11.8. F/P values of 3-10 are probably optimal forany particular IgG.

Protein concentration:

-   -   IgG (mg/ml)=[A(280)−0.31*A(495)]/1.4    -   IgM (mg/ml)=[A(280)−0.31*A(495)]/1.2

F/P ratio:

-   -   IgG: 3.1*A(495)/[A(280)−0.31*A(495)]    -   IgM: 15.9*A(495)/[A(280)−0.31*A(495)]        Detection by FRET Analysis

A bone marrow sample is obtained from an ALL patient and leukocytes areisolated according to standard procedures. Leukocytes are labeled withtwo cell surface markers to define a leukocyte subset viaimmunophenotypic characteristics. FITC-conjugated monoclonal anti-humanCD19 (FITC-CD19) and PE-conjugated monoclonal anti-human CD10 (PE-CD10)were used. Cells are then fixed according to standard procedures, e.g.in 1% paraformaldehyde, to preserve the integrity of the cell and itscontent. The cell membrane is permeabilized using a detergent such assaponin to make the cell interior accessible to probe set. Cells arelabeled for one hour at 4° C. in the dark with a mixture containing aprobe set according to the invention (0.1 to 0.3 microgram/ml of eachprobe), comprising a Cy3-labeled biotin-conjugated antibody against thehelix-loop-helix motif of TEL and a Cy5-labeled biotin-conjugatedantibody against the Runt domain of AML1. After washing of the cells toremove unbound probe, the cells are incubated with unlabeled avidin toinduce sufficiently close and stable juxtaposing of the two differentantibodies. The cells are then analyzed in a flow cytometer. Results areshown in FIG. 4. Panel A shows the evaluation of the TEL-AML1 fusionprotein in precursor-B-ALL cells obtained from a patient at the time ofdiagnosis. ALL blast cells are first gated on the basis of their lightscatter characteristics (forward scatter versus side scatter). Then, CD19-positive blast cells are gated (FL1 versus side scatter). Thepresence of the TEL-AML1 fusion protein is readily detectable in thesubset of CD19+/CD10+ ALL cells. In panel B, similar analyses are shownfrom the same patient after a five week therapy protocol to evaluate theeffectiveness of the treatment. Only 3% of the CD10+ blast cells ispositive for the TEL-AML1 fusion protein, i.e. only 0.2% of totalleukocytes. The detection of such a low frequency of TEL-AML1-positivecells (minimal residual disease) has not been shown before.

We have used the FaesCalibur to perform FRET measurements using Cy3 andCy5 as donor/acceptor pair. The 488 nm excitation is not optimal for Cy3(543 would be better), 632 is optimal for Cy5, and with this setup wecould obtain reasonable good FRET distribution curves (actually they arebetter than that obtained with FITC/TRITC pair because theautofluorescence is much less of a problem). In addition we could usethe 488->520 band for autofluorescence correction on a cell-by-cellbasis. Data acquisition and analysis were performed using Cell Quest Prosoftware.

REFERENCES

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1. A method for detecting a fusion protein's presence in a cell using aset of molecular probes comprising at least a first and a secondmolecular probe, each molecular probe able to recognize a binding sitepositioned at opposite sides of the fusion protein's fusion region, eachmolecular probe further directly conjugated to a dye wherein said dyestogether allow energy transfer, the first and the second molecularprobes directly conjugated to a reactive group that modulates themolecular probes' spatial organization after binding to a molecule ofinterest such that an increased likelihood of energy transfer existsbetween said dyes, wherein a reactive group of said first molecularprobe is not involved in binding to the molecule of interest and notdirectly reactive with said second molecular probe, said methodcomprising: providing the set of molecular probes, providing a samplecomprising a cell, contacting said sample with said set of probes underconditions that allow binding of said molecular probes directly to anysuch fusion protein within said sample, contacting said molecular probeswith a substance comprising at least two identical binding sites for thereactive group and able to link at least a reactive group of said firstmolecular probe to at least a reactive group of said second molecularprobe, and detecting juxtaposition of said molecular probes viafluorescence resonance energy transfer (FRET) to determine any suchfusion protein's presence.
 2. The method according to claim 1 whereinsaid substance causes said dyes to come within a distance of 2 to 100Ångstrom of each other.
 3. The method according to claim 1 wherein saidreactive group comprises biotin and wherein said substance comprisesavidin or streptavidin.